Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C(2)

Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function

Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 inSaccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle-(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translationin vivowith wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assemblyin vitroandin vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cel

Placeholder factors in ribosome biogenesis: please, pave my way

The synthesis of cytoplasmic eukaryotic ribosomes is an extraordinarily energy-demanding cellular activity that occurs progressively from the nucleolus to the cytoplasm. In the nucleolus, precursor rRNAs associate with a myriad of trans-acting factors and some ribosomal proteins to form pre-ribosomal particles. These factors include snoRNPs, nucleases, ATPases, GTPases, RNA helicases, and a vast list of proteins with no predicted enzymatic activity. Their coordinate activity orchestrates in a spatiotemporal manner the modification and processing of precursor rRNAs, the rearrangement reactions required for the formation of productive RNA folding intermediates, the ordered assembly of the ribosomal proteins, and the export of pre-ribosomal particles to the cytoplasm; thus, providing speed, directionality and accuracy to the overall process of formation of translation-competent ribosomes. Here, we review a particular class of trans-acting factors known as "placeholders". Placeholder factors temporarily bind selected ribosomal sites until these have achieved a structural context that is appropriate for exchanging the placeholder with another site-specific binding factor. By this strategy, placeholders sterically prevent premature recruitment of subsequently binding factors, premature formation of structures, avoid possible folding traps, and act as molecular clocks that supervise the correct progression of pre-ribosomal particles into functional ribosomal subunits. We summarize the current understanding of those factors that delay the assembly of distinct ribosomal proteins or subsequently bind key sites in pre-ribosomal particles. We also discuss recurrent examples of RNA-protein and protein-protein mimicry between rRNAs and/or factors, which have clear functional implications for the ribosome biogenesis pathway.España, MINECO BFU2013-42958-P and BFU2016-75352-

Yeast Polypeptide Exit Tunnel Ribosomal Proteins L17, L35, and L37 are Necessary to Recruit Late-assembling Factors Required for 27SB Pre-rRNA Processing

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2

Estudio funcional de proteínas ribosómicas durante la biogénesis de ribosomas en saccharomyces cerevisiae

Texto completo descargado desde TeseoEn el Capítulo I de esta tesis se muestran los resultados de la caracterización funcional de la proteína ribosómica L35, componente esencial de la subunidad grande del ribosoma. Nuestros resultados indican que la ausencia de L35 en las células tiene como consecuencia un déficit en la producción de subunidades 60S. Tal y como se demuestra por los experimentos de pulso-caza y de hibridación Northern, este déficit se debe a una fuerte inhibición en el procesamiento del pre-rRNA 27SB, dando lugar a niveles muy reducidos de los pre-rRNAs 7S y 25.5S y, en último término, de los rRNAS maduros 5.8S y 25S respectivamente. Además, se observa un claro retraso en el procesamiento temprano en los sitios A0, A1 y A2, que tiene como última consecuencia una reducción en los niveles del rRNA 18S y la aparición del precursor aberrante 23S. Estos resultados están en consonancia con estudios sistemáticos previos (1), donde L35 aparece en el grupo de las proteínas ribosómicas que intervienen en pasos intermedios del procesamiento de los pre-rRNAs. Nuestros resultados demuestran también que la proteína ribosómica L35 se asocia de forma estable a las partículas pre-60S tempranas y que su presencia es necesaria para el transporte óptimo de las partículas pre-60S del núcleo hacia el citoplasma. En el Capítulo II se presentan los resultados de la caracterización funcional de la proteína ribosómica L26, otro componente estructural de la subunidad grande del ribosoma. Estos resultados nos han permitido demostrar la no esencialidad de L26 en el crecimiento celular, durante el proceso de biogénesis de ribosomas y en la función ribosómica. Si bien, a través de los experimentos de pulso-caza se puede observar un ligero retraso en el procesamiento del pre-rRNA 27SB en ausencia de L26. Nuestros resultados demuestran también que la proteína ribosómica L26 se ensambla establemente en partículas pre-60S tempranas y su presencia es necesaria para un transporte eficiente de las partículas pre-60S desde el núcleo hacia el citoplasma. Globalmente, estos resultados establecen claras diferencias en la ruta de ensamblaje de la subunidad grande entre eucariotas y procariotas (3). Finalmente, en el Capítulo III se muestra el estudio de la dinámica de asociación y disociación de las partículas pre-60S del factor de actuación en trans Rlp7 y su relación con el ensamblaje de la proteína ribosómica L7. Rlp7 pertenece al grupo de factores de actuación en trans que se asemejan a proteínas ribosómicas, compartiendo un 40% de homología en sus secuencias. Se ha propuesto que el lugar de unión de estos factores a las partículas pre-ribosómicas es el mismo que ocupan las proteínas ribosómicas correspondientes en la partícula ribosómica madura (4, 5). Nuestros resultados confirman que Rlp7 se incorpora a partículas pre-ribosómicas 90S tempranas que contienen el pre-rRNA 35S y se libera de las partículas pre-ribosómicas tras el procesamiento del ITS2, en algún punto previo al procesamiento del extremo 3¿ del pre-rRNA 7S. Por su parte, la proteína ribosómica L7 se ensambla en el núcleo de forma estable a partículas pre-60S tempranas que contienen el pre-rRNA 27SA2. Ambas proteínas presentan dominios de unión a RNA en su secuencia. Nuestros resultados de CRAC revelan los lugares de unión de estas proteínas en el (pre-)rRNA. Rlp7 presenta tres lugares de unión al pre-rRNA en la zona del ITS2. La proteína ribosómica L7 no comparte ninguno de estos sitios de unión, mapeando exclusivamente en regiones del rRNA 25S. Nuestros experimentos de inmunoprecipitación de partículas (pre-)ribosómicas que contienen la proteína ribosómica L7 indican que Rlp7 está presente en estas partículas. A través de los experimentos de inmunoprecipitación de partículas pre-ribosómicas que contienen Rlp7, combinados con la técnica SILAC, se confirma la presencia de L7 en dichas partículas. Estos últimos resultados demuestran que ambas proteínas pueden coexistir en las mismas partículas pre-ribosómicas. En conjunto, estos resultados demuestran que el lugar de unión de Rlp7 a las partículas pre-60S no es el mismo que el de la proteína ribosómica L7 en las partículas 60S maduras y que no existe ninguna interdependencia entre estas proteínas durante el proceso de ensamblaje.Premio Extraordinario de Doctorado U

A model of energy deposition of energetic electrons and EUV emission in the Jovian and Saturnian atmospheres and implications

A model of the interaction between incident electron precipitation and H2 atmospheres is described. The local degraded primary and secondary electron energy distributions are calculated by using the continuous slowing down approximation. The altitude distribution of the ionization rate and various H and EUV H2 emissions are calculated for four different incident electron spectra. A total EUV H2 emission efficiency of 10.6 kR/incident erg/sq cm per sec is obtained for a pure H2 atmosphere. Comparison with the Voyager Jupiter observations indicates that an incident energy flux of about 8 ergs/sq cm per sec was present at the time of the encounter if the emission is located in an H2-dominated region. The local thermospheric heating rate was about 4 ergs/sq cm per sec for Jupiter and of the order of 0.1 erg/sq cm per sec for Saturn. A globally averaged atomic hydrogen production rate of about 1 x 10 to the 10th atoms/sq cm per sec is induced by the Jovian auroral electron precipitation, largely exceeding the solar EUV dissociation rate

Correction. Synthesis of dihydrothiophenes by an amino-directed thioisomünchnone-alkene cycloaddition reaction

The affiliation of one of the authors (M. E. Light) has been erroneously assigned. His current address is: Department of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, United Kingdo

Synthesis of dihydrothiophenes by an amino-directed thioisomünchnone-alkene cycloaddition reaction

Dihydrothiophenes can easily be obtained by a general protocol involving the reaction of 2-aminothioisomunchnones with electron-deficient alkenes. The overall process can be interpreted as a sequential [3+2] cycloaddition/ring-opening cyclization reaction. The structures of the products could be unequivocally assigned by X-ray diffraction analysis. A theoretical study at a semiempirical level (PM3) with a further single-point refinement of energies at the B3LYP/6-31G(*) level also offers mechanistic insights into the stereochemical outcome

Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles

International audienceRibosome biogenesis requires \textgreater300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation